Abstract The emergence of arthropod-borne viruses (arboviruses) is an ongoing public health concern. The conditions that lead to the emergence of new virus genotypes are poorly understood. In mosquitoes, temperature profoundly impacts virus transmission via replication rate and the likelihood that virus will disseminate from the midgut and ultimately be transmitted. Yet, there is a gap in the literature on the effect that extrinsic incubation (EI) temperature has on virus diversification in the mosquito. Therefore, we plan to study the role that temperature plays in arbovirus evolution during the EI period. First, we will determine the extent to which EI temperature impacts Zika virus (ZIKV) and West Nile virus (WNV) vector competence in Aedes and Culex mosquitoes respectively, and following this use RNA-seq to identify how the consensus genome changes in response to the temperature of EI. Next, we will assess temperature driven single-nucleotide variants and conduct population genetics analysis to determine the extent of the EI temperature?s effect on viral population diversity. This may provide a temperature specific model of viral evolution. Last, we will validate our temperature driven, viral evolution model by comparing it to locally transmitted WNV field isolates. The outcome of these studies will provide insight to arbovirus evolution and potentially be used to predict environmental conditions that may lead to evolution and selection of novel virus genotypes that cause severe outbreaks. Hypothesis: We hypothesis that when arboviruses such as WNV and ZIKV undergo extrinsic incubation in their respective vector, a high temperature of incubation drives viral population diversity, leading to mutant haplotypes being transmitted. This hypothesis will be tested in three specific aims Aim 1. Determine the extent in which the temperature of extrinsic incubation impacts ZIKV and WNV vector competence in mosquitoes and identify any temperature driven consistent and predictable consensus genome changes. Aim 2. Determine how the temperature of extrinsic incubation influences ZIKV and WNV population diversity within mosquitoes. Aim 3. Characterize the effects of temperature on WNV evolution in Fort Collins from 2013-2017.